Vaccinia virus replicates solely within the cytoplasm of the infected cell, displaying significant genetic and physical autonomy. Among its approximately 200 genes are those encoding the F10 protein kinase, which plays a prime role in the initiation and regulation of virion morphogenesis, and the H1 protein phosphatase, which is required to establish the structural integrity and transcriptional competence of nascent virions. The current proposal reflects our ongoing interest in the structure and function of the F10 kinase, and of various viral phosphoproteins which participate in the biogenesis of the virion membrane and the formation and maturation of the virion core. This process, in concert with the viral H1 phosphatase, culminates with the formation of stable, transcriptionally competent, and infectious virions. The two aims are: Aim 1: Analysis of the structure and function of the F10 kinase. A. How is the catalytic activity and intracellular localization of F10 regulated in vivo? B. Which enzyme/substrate and protein/protein interactions are involved in the regulation of virion morphogenesis by F10? Aim II: Phosphorylation and morphogenesis: understanding the origins of the virion membrane and analyzing the structure and function of viral phosphoproteins involved in membrane biogenesis, virion maturation, and core formation. A. What is the origin of the membrane precursors? B. How do the membrane phosphoproteins A14 and A17 interact with each other and with additional proteins? C. What is the role of the A13 protein, another phosphorylated component of the virion membrane, in recruiting other membrane proteins and in enabling the maturation of immature virions? D. What is the role of the F18 and A5 phosphoproteins in the assembly of a stable and transcriptionally competent core, and how is this role modulated by phosphorylation and dephosphorylation? In addition to elucidating problems of broad cell biological significance, these problems are of increasing biomedical importance. Although variola virus, the causative agent of smallpox, has been eradicated as a natural threat, there is significant concern about its possible use as an agent of bioterrorism. A deeper understanding of the life cycle of vaccinia virus, which has been used so effectively as the vaccine for protection against smallpox, will provide a solid foundation for the development of rational anti-poxvirus therapies and attenuated vaccines.